The broad objective of this project is to analyze at the molecular level the regulatory mechanisms of gene expression controlled by double-stranded RNA (dsRNA) in human cells. Molecules of dsRNA are either produced by viral infection or transposon activity, or they are expressed in a regulated manner from cellular genes that encode short inverted repeat sequences (microRNAs). Introduction of dsRNA into cells cognate to cellular genes has revolutionized cell biological studies enabling investigators to suppress their favorite gene for functional analysis and has opened the doors to new ways of gene-specific therapy. The aim of this project is to identify and characterize the protein components involved in RNA silencing and to understand at molecular level how silencing-active ribonucleoprotein complexes are formed and exert their function. 1. Characterization of the human siRNA/miRNA-protein complexes. 1.1 Investigate the role of siRNA/miRNA-associated Argonaute protein members (elF2C family, HIWI, HILl) and define their interaction network with cellular components required for RNAi and miRNA-mediated gene regulation. 1.2 Reconstitute the sequence-specific endonuclease complex (RISC) from recombinant proteins and short duplex or single-stranded siRNA molecules. 2. Isolation of miRNP/target mRNA complexes and identification of the cellular mRNAs subjected to microRNA-mediated control.